This Phase 2 STTR application will allow the Foundation for Applied Molecular Evolution (FfAME) to complete its transfer of technology to Firebird Biomolecular Sciences LLC, allowing Firebird to commercialize nucleoside triphosphates where the 3'-OH groups are blocked by a 3'-ONH2 group. Triphosphates having this modification are accepted by DNA polymerases and other enzymes. After being incorporated at the 3'-end of a growing oligonucleotide, the 3'-ONH2 group blocks extension (just as a 2',3'-dideoxynucleotide does). However, this blockage is reversible;buffered sodium nitrite rapidly cleaves the O-NH2 bond to generate the natural 3'-OH group, yielding under mild conditions a primer that can again be extended by DNA polymerase. Further, the 3'-ONH2 group offers a handle that allows the terminated DNA to selectively react with certain functional groups, allowing for the recovery of the extended, terminated DNA from complex biological mixtures. This is useful in many architectures that analyze DNA, including "next generation" sequencing that promises to personalize diagnostics and therapeutics, tools to detect single nucleotide polymorphisms, strategies to do "effectiveness research" for medical treatments, and other architectures that have increasing complexity and risk. Therefore, the products to be developed here have multiple applications in a variety of research settings, genetic disease discovery, and human diagnostics. This Phase 2 project, together with Firebird's internal funding, will build a production pipeline to prepare 12 different triphosphates carrying the reversible terminator, as well as four phosphoramidites to support kits that use these. The synthetic chemistry developed in the Phase 1 period will support this pipeline. Further, tools used in the Phase I period to develop polymerases to handle unnatural nucleoside triphosphates will be applied to create still better polymerases for assays that exploit our triphosphates. Finally, kits for DNA analysis will be produced that exploit these reversible terminators, in many cases with other reagent innovations that are proprietary to FfAME, Firebird, and others. Several customers are already exploiting these compounds. These include foreign customers, whose purchase of these reagents is creating American jobs through the sale of products internationally. Growing its portfolio of nucleic acid analogs underlies the next phase in the development of Firebird as a corporation. PUBLIC HEALTH RELEVANCE: Firebird Biomolecular Sciences LLC commercializes innovative reagents, polymerases that exploit them, and assays and architectures based on them, for the detection, analysis and sequencing of nucleic acids. Firebird also develops software to exploit genomic sequence data, and what it means in biology and medicine. This Phase 2 STTR project will create over two dozen jobs through the development of a series of reversible terminators suitable for next generation DNA sequencing, SNP detection, and DNA capture.